Bacterial pathogens have developed mechanisms of resistance to antibiotics, reducing or eliminating drastically the effectiveness of antibacterial drugs. Multi-drug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, which are difficult to reach for antibiotics and immune cells. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we synthesized a small library of N-substituted hydroxylamine (N-HA) compounds with antibacterial activity. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria by inhibiting the bacterial ribonucleotide reductase (RNR) enzyme, which provides the building blocks for DNA synthesis and repair. Furthermore, the most promising compounds are able to reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.