This study investigates the influence of pump laser fluence on ultrafast structural dynamics of carboxymyoglobin (MbCO) using time-resolved serial femtosecond crystallography (TR-SFX). The research highlights that different pump laser fluences significantly affect the structural changes and coherent oscillations of the Fe–CO bond distance in MbCO. The results show that high pump laser fluence leads to multiphoton absorption, altering the protein response and potentially affecting the validity of conclusions drawn about biologically relevant single-photon-induced reactions. The study confirms the feasibility and necessity of performing ultrafast TR-SFX experiments in the linear photoexcitation regime to ensure mechanistically relevant insights. The study demonstrates that the dynamics of structural changes and the observed coherent oscillations of the Fe–CO bond distance depend strongly on pump laser energy, consistent with quantum chemical analysis. The results suggest that high fluence experiments may not accurately represent single-photon-induced reactions, as multiphoton effects can lead to different photodissociation mechanisms. The research also shows that the occupancy of the photodissociated state increases with laser fluence, but the structural changes observed are influenced by both occupancy and multiphoton absorption. The study reveals that the Fe–CO bond distance and other structural features change with different laser fluences, with the lowest fluence showing oscillations and the highest fluence showing less pronounced changes. The findings indicate that the photophysical mechanism of CO dissociation differs between single and two-photon absorption, leading to different structural dynamics. The results emphasize the importance of using appropriate photoexcitation conditions in TR-SFX experiments to ensure accurate mechanistic insights. The study also highlights the need for further research to understand the effects of multiphoton absorption on structural dynamics and to refine the interpretation of TR-SFX data.This study investigates the influence of pump laser fluence on ultrafast structural dynamics of carboxymyoglobin (MbCO) using time-resolved serial femtosecond crystallography (TR-SFX). The research highlights that different pump laser fluences significantly affect the structural changes and coherent oscillations of the Fe–CO bond distance in MbCO. The results show that high pump laser fluence leads to multiphoton absorption, altering the protein response and potentially affecting the validity of conclusions drawn about biologically relevant single-photon-induced reactions. The study confirms the feasibility and necessity of performing ultrafast TR-SFX experiments in the linear photoexcitation regime to ensure mechanistically relevant insights. The study demonstrates that the dynamics of structural changes and the observed coherent oscillations of the Fe–CO bond distance depend strongly on pump laser energy, consistent with quantum chemical analysis. The results suggest that high fluence experiments may not accurately represent single-photon-induced reactions, as multiphoton effects can lead to different photodissociation mechanisms. The research also shows that the occupancy of the photodissociated state increases with laser fluence, but the structural changes observed are influenced by both occupancy and multiphoton absorption. The study reveals that the Fe–CO bond distance and other structural features change with different laser fluences, with the lowest fluence showing oscillations and the highest fluence showing less pronounced changes. The findings indicate that the photophysical mechanism of CO dissociation differs between single and two-photon absorption, leading to different structural dynamics. The results emphasize the importance of using appropriate photoexcitation conditions in TR-SFX experiments to ensure accurate mechanistic insights. The study also highlights the need for further research to understand the effects of multiphoton absorption on structural dynamics and to refine the interpretation of TR-SFX data.