This study aimed to develop a hydroxyethyl cellulose-based topical formulation containing probiotics and to evaluate its antimicrobial action using in vivo and ex vivo models. Initially, the antagonistic effects of ATCC 10863, ATCC 23271, ATCC 8014 and LP-G18-A11 were analyzed against ATCC 29212, ATCC 700603, ATCC 27853 and ATCC 2785. The best action was seen for LP-G18-A11, which presented high inhibition against and . Then, lactobacilli strains were incorporated into hydroxyethyl cellulose-based gels (natrosol); however, only the LP-G18-A11-incorporated gels (5% and 3%) showed antimicrobial effects. The LP-G18-A11 gel (5%) maintained its antimicrobial effects and viability up to 14 and 90 days at 25 °C and 4 °C, respectively. In the ex vivo assay using porcine skin, the LP-G18-A11 gel (5%) significantly reduced the skin loads of and after 24 h, while only was reduced after 72 h. Moreover, the LP-G18-A11 gel (5%) showed stability in the preliminary and accelerated assays. Taken together, the results show the antimicrobial potential of LP-G18-A11, which may be applied in the development of new dressings for the treatment of infected wounds.