ABSTRACT In two pairs of clinical colistin-susceptible/colistin-resistant (Cst ^s /Cst ^r ) Acinetobacter baumannii strains, the Cst ^r strains showed significantly decreased biofilm formation in static and dynamic assays ( P < 0.001) and lower relative fitness ( P < 0.05) compared with those of the Cst ^s counterparts. The whole-genome sequencing comparison of strain pairs identified a mutation converting a stop codon to lysine (*241K) in LpsB (involved in lipopolysaccharide [LPS] synthesis) in one Cst ^r strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other.