Small antimicrobial β2,2-amino acid derivatives (Mw < 500 Da) are reported to display high antibacterial activity against suspended Gram-positive strains combined with low hemolytic activity. In the present study, the anti-biofilm activity of six β2,2-amino acid derivatives (A1–A6) against Staphylococcus aureus (ATCC 25923) was investigated. The derivatives displayed IC50 values between 5.4 and 42.8 μM for inhibition of biofilm formation, and concentrations between 22.4 and 38.4 μM had substantial effects on preformed biofilms. The lead derivative A2 showed high killing capacity (log R), and it caused distinct ultrastructural changes in the biofilms as shown by electron and atomic force microscopy. The anti-biofilm properties of A2 was preserved under high salinity conditions. Extended screening showed also high activity of A2 against Escherichia coli (XL1 Blue) biofilms. These advantageous features together with high activity against preformed biofilms make β2,2-amino acid derivatives a promising class of compounds for further development of anti-biofilm agents.