Non-antibiotics and antibiotics use distinct toxicity mechanisms in bacteria but are both affected by shared efflux systems. A study analyzed the antibacterial activity of 200 drugs using genetic screens with thousands of barcoded Escherichia coli knockouts, revealing that non-antibiotics operate through mechanisms orthogonal to standard antibiotics. Network analysis showed antibiotics clustered by their mode of action, while non-antibiotics remained unconnected, suggesting shared but unexploited targets. Efflux systems impact both antibiotics and non-antibiotics, highlighting the need to investigate their effects on antibiotic cross-resistance. Genetic screens identified that non-antibiotics target different pathways than antibiotics, with some sharing common toxicity mechanisms. Analysis of drug transport systems showed that inactivating these systems increased bacterial sensitivity to both antibiotics and non-antibiotics. Evolution experiments demonstrated that resistance to non-antibiotics can lead to multi-drug resistance and antibiotic cross-resistance. The study underscores the importance of understanding non-antibiotic toxicity mechanisms to identify new antimicrobial targets and mitigate unintended resistance. The findings suggest that non-antibiotics may target unexploited bacterial pathways and that shared efflux systems could contribute to cross-resistance. The research highlights the need for further studies to understand the impact of non-antibiotics on the human microbiome and antibiotic efficacy.Non-antibiotics and antibiotics use distinct toxicity mechanisms in bacteria but are both affected by shared efflux systems. A study analyzed the antibacterial activity of 200 drugs using genetic screens with thousands of barcoded Escherichia coli knockouts, revealing that non-antibiotics operate through mechanisms orthogonal to standard antibiotics. Network analysis showed antibiotics clustered by their mode of action, while non-antibiotics remained unconnected, suggesting shared but unexploited targets. Efflux systems impact both antibiotics and non-antibiotics, highlighting the need to investigate their effects on antibiotic cross-resistance. Genetic screens identified that non-antibiotics target different pathways than antibiotics, with some sharing common toxicity mechanisms. Analysis of drug transport systems showed that inactivating these systems increased bacterial sensitivity to both antibiotics and non-antibiotics. Evolution experiments demonstrated that resistance to non-antibiotics can lead to multi-drug resistance and antibiotic cross-resistance. The study underscores the importance of understanding non-antibiotic toxicity mechanisms to identify new antimicrobial targets and mitigate unintended resistance. The findings suggest that non-antibiotics may target unexploited bacterial pathways and that shared efflux systems could contribute to cross-resistance. The research highlights the need for further studies to understand the impact of non-antibiotics on the human microbiome and antibiotic efficacy.