This study investigates the influence of pump laser fluence on the ultrafast structural dynamics of carboxymyoglobin (MbCO) using time-resolved serial femtosecond crystallography (TR-SFX). The research highlights that different pump laser fluences lead to significantly different results, particularly in the dynamics of structural changes and the observed indicators of coherent oscillations in the Fe–CO bond distance. These findings are consistent with quantum chemical analysis, suggesting that the photodissociation mechanism depends strongly on the pump laser energy. The study confirms the feasibility and necessity of performing ultrafast TR-SFX pump–probe experiments in the linear photoexcitation regime, which is crucial for obtaining mechanistically relevant insights into biological systems. The results also emphasize the importance of careful consideration of photoexcitation conditions to ensure that the experimental approach accurately reflects biologically relevant single-photon-induced reactions.This study investigates the influence of pump laser fluence on the ultrafast structural dynamics of carboxymyoglobin (MbCO) using time-resolved serial femtosecond crystallography (TR-SFX). The research highlights that different pump laser fluences lead to significantly different results, particularly in the dynamics of structural changes and the observed indicators of coherent oscillations in the Fe–CO bond distance. These findings are consistent with quantum chemical analysis, suggesting that the photodissociation mechanism depends strongly on the pump laser energy. The study confirms the feasibility and necessity of performing ultrafast TR-SFX pump–probe experiments in the linear photoexcitation regime, which is crucial for obtaining mechanistically relevant insights into biological systems. The results also emphasize the importance of careful consideration of photoexcitation conditions to ensure that the experimental approach accurately reflects biologically relevant single-photon-induced reactions.