Pathogenic, antibiotic-resistant, and biofilm-forming bacteria can be transferred to humans through the consumption of contaminated seafood. The present study was carried out to determine antibiotic resistance profiles and virulence determinants in biofilm-forming isolated from seafood in Bangladesh. A total of 150 seafood samples, including shrimp (n = 50), crabs (n = 25), and marine fish (n = 75), were screened using cultural, staining, biochemical, polymerase chain reaction (PCR), Congo red (CR), and disk diffusion (DD) assays. In PCR, was detected in 27.3% (41/150; CI 20.8; 34.9) of samples, where marine fish (34.7%, CI 24.9; 45.9) had the highest prevalence ( < 0.05) compared to crabs (32%, CI 17.2; 51.6) and shrimp (14%, CI 7.0; 26.1). Thirty-two (78.1%, CI 63.3; 88.0) of the isolates were determined to be biofilm formers in the CR test, where 43.9% (18/41, CI 29.9; 59.0) and 34.2% (14/41, CI 21.6; 49.5) of the isolates were strong and intermediate biofilm formers, respectively. In PCR, virulence genes, i.e., (100%), (92.7%), (68.3%), (65.9%), (63.4%), (53.7%), (51.2%), and (43.9%), were detected in isolates. All the isolates were phenotypically resistant to ≥3 antimicrobial categories and ≥3 antibiotics, including WHO-classified reserve antibiotics linezolid (70.7%) and fosfomycin (19.5%). Moreover, the multiple antibiotic resistance index ranged up to 0.8, showing resistance to ten antibiotics and eight antibiotic classes. In this study, the prevalence of virulence genes and antibiotic resistance was significantly greater ( < 0.05) in strong biofilm-forming strains as compared to strains with intermediate and non-biofilm-forming abilities. As far as we know, this study, for the first time in Bangladesh, determined antibiotic resistance and detected virulence genes in biofilm-forming isolated from seafood samples. The data from this study could play a significant role in evaluating potential health hazards linked to the ingestion of uncooked or minimally processed seafood.