In the alarming context of antibiotic resistance, we explored the antibacterial potential of French mushrooms against wild-type and multidrug-resistant (MDR) bacteria. In order to accelerate the discovery of promising compounds, screenings were carried out by TLC-direct bioautography. A total of 70 extracts from 31 mushroom species were evaluated against five wild-type bacteria: Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Pseudomonas aeruginosa. This first screening revealed that 95% of the extracts contained antibacterial compounds. Generally, it was observed that EtOAc extracts exhibited more active compounds than methanolic extracts. In addition, all extracts were overall more active against Gram-positive bacteria than against Gram-negative strains. The most promising mushroom extracts were then screened against various multidrug-resistant strains of S. aureus and E. coli. Activity was globally less on MDR strains; however, two mushroom species, Fomitopsis pinicola and Scleroderma citrinum, still contained several compounds inhibiting the growth of these MDR pathogenic bacteria. Stearic acid was identified as a ubiquitous compound contributing to the antibacterial defence of mushrooms. This screening revealed the potential of macromycetes as a source of antibacterial compounds; further assays are necessary to consider fungal compounds as promising drugs to counter antibiotic resistance.