The study investigates the antibacterial activity of non-antibiotic drugs and their impact on the human microbiome. Using genetic screens with *Escherichia coli* knockouts, the researchers identified 176 non-antibiotics and 142 antibiotics that inhibit bacterial growth. Network analysis revealed that antibiotics cluster into modules based on their mode of action, while most non-antibiotics remain unconnected, suggesting they operate through different toxicity mechanisms. The study also found that efflux systems significantly impact both antibiotics and non-antibiotics, indicating that resistance to non-antibiotics can lead to cross-resistance to antibiotics. Evolution experiments with three non-antibiotics confirmed these findings, demonstrating that evolved resistance can result in broad antibiotic cross-resistance and uncover novel cellular targets. The results highlight the need to investigate non-antibiotic drugs for their potential as antimicrobials and the potential risks of unintended cross-resistance.The study investigates the antibacterial activity of non-antibiotic drugs and their impact on the human microbiome. Using genetic screens with *Escherichia coli* knockouts, the researchers identified 176 non-antibiotics and 142 antibiotics that inhibit bacterial growth. Network analysis revealed that antibiotics cluster into modules based on their mode of action, while most non-antibiotics remain unconnected, suggesting they operate through different toxicity mechanisms. The study also found that efflux systems significantly impact both antibiotics and non-antibiotics, indicating that resistance to non-antibiotics can lead to cross-resistance to antibiotics. Evolution experiments with three non-antibiotics confirmed these findings, demonstrating that evolved resistance can result in broad antibiotic cross-resistance and uncover novel cellular targets. The results highlight the need to investigate non-antibiotic drugs for their potential as antimicrobials and the potential risks of unintended cross-resistance.