We prepared non-woven electrospun polylactic acid (PLA) membranes containing functionalized sepiolite fibrillar particles (5 wt%). Biocidal activity was achieved by functionalizing sepiolite fibers with Ag (26 wt%) and Cu (26 wt%), which were embedded in the fiber surface. Sepiolite particles were negatively charged in all cases, with zeta potential in the range of −15 to −37 mV, increasing their negative charge with pH. The membranes were biofouled in a cross-flow filtration device using model biofoulants, cultures of Saccharomyces cerevisiae (pH 4.5) and Pseudomonas putida (pH 7.5), which were pumped across the membrane for 24/48 h. The assessment of active biomass was performed by measuring the concentration of adenosine-5′-triphosphate (ATP) in used membranes. The results showed a significant decrease in the amount of surface ATP for PLA loaded with Ag and Cu functionalized sepiolite. The effect was particularly intense for Ag-sepiolite in contact with Saccharomyces cerevisiae, where the reduction amounted to 85% compared to neat PLA (control). This was attributed to the differences in ion availability as a function of pH and to the presence of chloride ions in solution. Pristine sepiolite (without functionalization) did not show any noticeable effect.