In the majority of Latin-American countries, including Argentina, there is a limited availability of vaginal bioproducts containing probiotics in the market. In addition, the conventional treatments of genital tract infections in women represent a high cost to the public health systems. The future development of this type of bioproducts that employ specific lactobacilli strains would not only have a meaningful impact on women's health but would also represent a significant challenge to the pharmaceutical industry. The aims of the work described in this paper were (i) to study different pharmaceutical formulations of vaginal ovules containing Lactobacillus fermentum L23 and L. rhamnosus L60, to determine in which formulation lactobacilli viability was sustained for longer time and (ii) to evaluate if probiotic strains maintained both the antimicrobial activity and biofilm-producing ability after being recovered from the ovules. In this study, we developed and characterized three pharmaceutical formulations containing different glycerol amounts and specific lactobacilli strains. Three relevant parameters, cell viability, antimicrobial activity, and biofilm production, by lactobacilli recovered from the ovules were tested. Although the viability of L23 and L60 strains was mainly influenced by high ovule's glycerol proportion, they survived at 4 °C during the 180 days. Both lactobacilli's antimicrobial activity and biofilm-producing ability were maintained for all treatments. In conclusion, employing a much reduced number of components, we were able to select the most suitable pharmaceutical formulation which maintained not only lactobacilli viability for a long period of time but also their antimicrobial activity and biofilm-producing ability.