The aim of this study was to evaluate the phenotypic and molecular patterns of biofilm formation in infection and colonization isolates of Acinetobacter spp. from patients who were admitted in a public hospital of Recife-PE-Brazil in 2018-2019. For the biofilm phenotypic analysis, Acinetobacter spp. isolates were evaluated by the crystal violet staining method; the search of virulence genes (bap, ompA, epsA, csuE and bfmS) was performed by PCR; and the ERIC-PCR was performed for molecular typing. Amongst the 38 Acinetobacter spp. isolates, 20 were isolated from infections and 18 from colonization. The resistance profile pointed that 86.85% (33/38) of the isolates were multidrug-resistant, being three infection isolates, and two colonization isolates resistant to polymyxin B. All the isolates were able to produce biofilm and they had at least one of the investigated virulence genes on their molecular profile, but the bap gene was found in 100% of them. No clones were detected by ERIC-PCR. There was no correlation between biofilm formation and the resistance profile of the bacteria, neither to the molecular profile of the virulence genes. Thus, the ability of Acinetobacter spp. to form biofilm is probably related to the high frequency of virulence genes.