The objective of this study was to demonstrate the effectiveness of disinfection procedures to reduce bacterial load of rainbow trout, Oncorhynchus mykiss (Walbaum 1792), semen. Fresh semen was obtained from 3-4-year-old male species by abdominal sampling of sperm into pre-cooled test tubes. After sperm cryopreservation and thawing, experiments were accomplished at 4-9 degrees C. 'Swim-up' and gradient centrifugation were used as a sperm washing method with commercial kits. Phosphate buffered saline was also used as washing solution. Bacterial growth tests were employed before and after washing the semen samples. Samples were inoculated on tryptic soy agar (TSA), modified Anacker and Ordal agar (MAOA) as well as brain heart infusion (BHI) agar. After using 'swim-up' method for washing the semen, many bacterial colonies were observed. However, after semen washing with gradient centrifugation, lower bacterial growth was observed on TSA, MAOA and BHI. Some motile (40%) spermatozoa were obtained doing gradient washing procedure. Although sperm motility was not satisfactory, apparently the gradient centrifugation method reduced bacterial contamination as known from the mammalians.