Acinetobacter baumannii (A. baumannii) is considered a critical human pathogen due to multi-drug resistance and increased infections. As a result of the resistance of A. baumannii biofilms to antimicrobial agents, it is necessary to develop new biofilm control strategies. In the present study, we evaluated the efficacy of two previously isolated bacteriophage C2 phage, K3 phage and phage cocktail (C2 + K3 phage) as a therapeutic agent in combination with antibiotic (colistin) against biofilm of multidrug-resistant A. baumannii strains (n = 24). The effects of phage and antibiotics on mature biofilm were investigated simultaneously and sequentially in 24 and 48 h. The combination protocol was more effective than antibiotics alone in 54.16% of the strains in 24 h. The sequential application was more effective than the simultaneous protocol compared with the 24 h single applications. When the application of antibiotics and phages alone was compared with their combined administration in 48 h. The sequential and simultaneous applications were more effective than single applications in all strains except two. We observed that combination of phage and antibiotics could increase biofilm eradication and provides new insights into the use of bacteriophages and antibiotics in the treatment of biofilm-associated infections caused by antibiotic-resistant bacteria.