The aim of this work was to co‐nanoencapsulate Lactobacillus acidophilus (LCFE) and Bifidobacterium bifidum (BCFE) cell‐free extract and zenyan (Carum copticum L.) seed water (ZWE) and ethanolic (ZEE) extract in electrospun cellulose acetate (CA) nanofibers and evaluate antimicrobial potential. The zeta potential, SEM image, antibacterial (MIC and MBC), and antifungal (MIC and MFC) activities were evaluated. TPC (total phenol content) of water and ethanol extract of zenyan seed were 14.05 and 136.44 mg GAE/g, respectively. A zeta potential of −40.25, −45.80, −43.71, 48.55, 35.50, 47.93, 31.50, 44.69, and −29.61 mV was found for nanofibers of pure CA (cellulose acetate), CA/LCFE, CA/BCFE, CA/ZWE, CA/ZEE, CA/LCFE/ZWE, CA/LCFE/ZEE, CA/BCFE/ZWE, and CA/LCFE/ZEE, respectively. CA electrospun nanofiber loaded with different extracts showed nanosized diameter and uniform structure. Nanoencapsulated extracts showed considerably higher antibacterial and antifungal activity compared to free extracts. Antibacterial activity of lactobacilli cell‐free extract was higher than bifidobacteria, which indicated the presence of the higher amount of antibacterial compounds in lactobacilli extract. Gram‐positive bacteria (S. aureus and L. monocytogenes) had the lowest MIC and MBC of free and nanoencapsulated extracts while Gram‐negatives (E. coli, S. dysenteriae, and S. enteritidis) had higher MIC and MBC. CA‐coated zenyan extracts (water and ethanolic) inhibited the growth of the assayed fungi at the MIC ranging 0.25 to 0.95%. These concentrations were 1.5–2 times lower than those obtained for pure extracts. For nanoencapsulated cell‐free extracts of both probiotics, the MIC values were about five times lower than the free extracts. The highest antimicrobial activity obtained for CA nanofibers contained zenyan ethanolic extract and cell‐free extract of lactobacilli or bifidobacteria.