Bacteriocins are known for anti-microbial properties against various pathogens. In the present study, bacteriocin ST22Ch produced by Lactobacillus sakei isolated from salpicao, a traditional pork product from the northwest of Portugal, inhibited the growth of Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Lactobacillus rhamnosus, Listeria ivanovii subsp. ivanovii, Listeira monocytogenes, Pseudomonas spp., Staphylococcus aureus, Streptococcus caprinus and Streptococcus spp. In particular, low levels of bacteriocin ST22Ch activity (approximately 200 AU/mL) was detected after 3 h of growth in MRS broth. Maximal production (1600 AU/mL) of bacteriocin ST22Ch was recorded after 19 h in MRS broth, stay constant only for 5 h and decrease to 800 AU/mL and only when incubated at 30 °C. During 35 h of growth, the optical density increased from 0.15 to 5.73. Highest bacteriocin ST22Ch production (3200 AU/mL) was records in presence of yeast extract as single organic nitrogen source (20.0 g/L). Presence of tryptone (20.0 g/L) or combination of tryptone and meat extract (12.5 g/L and 7.5 g/L) or yeast extract and meat extract (10.0 g/L and 10.0 g/L, respectively) yielded 1600 AU/mL bacteriocin production. Presence of glycerol at 1.0 g/L to 5.0 g/L not effect the bacteriocin ST22Ch production. Tween 80 added to the MRS medium (5.0 g/L) increase bacteriocin ST22Ch production up to 6400 AU/mL. Exclusion of manganese sulphate results in reduction of activity in the cell-free supernatant. Exclusion of magnesium sulphate from the media formula increases the bacteriocin production to 3200 AU/mL.