One hundred and thirty-seven lactic acid bacteria (LAB), previously isolated from wheat (Triticum durum) grains and non-conventional flour samples, were tested for the production of antibacterial substances. A total of 16 strains (5 Enterococcus faecium, 5 Enterococcus mundtii, 4 Pediococcus pentosaceus, 1 Lactobacillus coryniformis and 1 Lactococcus garvieae) were found to inhibit the growth of Listeria innocua. The antibacterial activities were preliminarily investigated for their general behaviour with proteolytic (proteinase K, protease B and trypsin), amylolytic (-amylase) and lipolytic (lipase) enzymes, after heat treatment, and exposure to different pHs and ethanol concentrations. Bacteriocin-like inhibitory substances (BLIS) were also characterized for their inhibition spectra against non-pathogenic and pathogenic food-associated and human pathogenic bacteria. LAB showing the best characteristics in terms of inhibition spectrum, inhibition activity and mode of action (bactericidal) belonged to the species Ent. mundtii. The high percentage (11.68%) of BLIS-producing strains detected confirmed previous observations that raw materials may harbour higher numbers of bacteriocinogenic LAB than fermented foods.