Obesity in the postmenopausal period is associated with an increased risk of cardiovascular diseases in women. One of the key drivers of cardiovascular risk is endothelial dysfunction; thus, this is also a crucial point for studies on new therapeutic methods of cardioprotective properties. The aim of the current study was to evaluate the effect of two doses of multispecies probiotic Ecologic® Barrier supplement on functional (primary endpoint) and biochemical parameters (secondary endpoint) of endothelial dysfunction in obese postmenopausal women in a 12-week randomized, placebo-controlled clinical trial. A total of 81 obese Caucasian women participated in the trial. The subjects were randomly assigned to three groups that received a placebo, a low dose (LD) (2.5 × 109 colony forming units (CFU) per day), or a high dose (HD) (1 × 1010 CFU per day) of lyophilisate powder containing live multispecies probiotic bacteria. The probiotic supplement was administered each day for 12 weeks in two equal portions. A high dose probiotic supplementation for 12 weeks decreased systolic blood pressure, vascular endothelial growth factor, pulse wave analysis systolic pressure, pulse wave analysis pulse pressure, pulse wave analysis augmentation index, pulse wave velocity, interleukin-6, tumor necrosis factor alpha, and thrombomodulin. Low doses of probiotic supplementation decreased the systolic blood pressure and interleukin-6 levels. The mean changes in the estimated parameters, compared among the three groups, revealed significant differences in the vascular endothelial growth factor, the pulse wave analysis systolic pressure, the pulse wave analysis augmentation index, the pulse wave velocity, the tumor necrosis factor alpha, and thrombomodulin. The post hoc tests showed significant differences for all parameters between HD and the placebo group, and HD and LD (besides pulse wave analysis augmentation index). We show for the first time that supplementation with multispecies probiotic Ecologic® Barrier favorably modifies both functional and biochemical markers of vascular dysfunction in obese postmenopausal women.