Antimicrobial resistance studies have focused on clinical bacteria, neglecting the role of resistant isolates in natural environments. However, oceans are daily contaminated with high loads of antimicrobials and resistant bacteria from agro-industrial and urban activities. Deep-sea sediment is a challenging environment that may select microbial strains with resistance to chemicals and ability to form biofilms, becoming a potential reservoir of resistance genes. We evaluated the susceptibility to antimicrobials of six Pseudomonas sp., five Bacillus sp., two Brevibacillus sp. and two Paenibacillus sp. from deep-sea sediments of the Pelotas Basin (Brazil) by the disk diffusion and microdilution tests. Pseudomonas and Bacillales were tested against 11 and 7 antimicrobials, respectively. Biofilms of susceptible isolates were exposed to antimicrobials to determine the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC). All Pseudomonas were resistant to aztreonam at very high concentrations (up to 2048 μg/mL). MBIC values were significantly higher than respective MICs, and only one third of biofilms were eradicated. These results underscore the importance of the study, as one of the first reporting antimicrobial tolerance of biofilms of cultivable bacteria from deep-sea sediments, contributing to the knowledge of bacterial resistance in these environments, concerning One Health issues.