Seventeen Leuconostoc mesenteroides and 33 Lactobacillus paracasei subsp. paracasei from traditional Pecorino di Filiano cheese were tested for their potential use as adjunct starters, and a study of their genetic variability was carried out. Forty one per cent (41%) of Leu. mesenteroides and 46% of Lb. paracasei subsp. paracasei showed medium–high proteolytic activity, while high lipolytic activity was detected in 18% of Lb. paracasei subsp. paracasei. The aminopeptidase activity of Lb. paracasei subsp. paracasei was higher than that of Leu. mesenteroides. Strain diversity by RAPD analysis showed a high degree of heterogeneity. This study identified strains with unusual properties that could be good candidates as adjuncts in a starter to manufacture PF cheese.