Intestinal bacteria in marine fish may produce antimicrobial substances which inhibit pathogenic bacteria. The aim of this study was to determine the influence of a change of fish diet on the antimicrobial activity of the culturable aerobic gut microflora of Senegalese sole, Solea senegalensis. Pre-adult 15-month-old fish previously fed on an artificial diet, were fed polychaetes (Hediste diversicolor), which form part of the natural diet of Senegalese sole. Samples were taken0, 3 and 6 weeks after start of the experiment from the stomach, small and large intestine of the fish. The bacterial strains isolated from these samples were sub-cultured to pure cultures and stored at −80°C. Several biochemical tests were run to obtain some basic phenotypic characteristics of the isolated strains. Amplification and sequencing of 16S rDNA fragments were used to identify the majority of the bacterial strains isolated. The identification by use of this molecular approach gave results in agreement to the phenotypic characterization. Feeding with polychaetes significantly increased (P<0.05) the numbers of presumptive Vibrio isolates in the gut. The number of bacterial strains with antimicrobial activity, as determined by two in vitro approaches, was significantly (P<0.05) increased by feeding with polychaetes.