Amidst the rising popularity of craft beers, it would be opportune to develop a novel, unfiltered and unpasteurized sour beer with high probiotic live counts. However, as beer typically contains hop iso-α-acids that prevent the growth and survival of probiotic lactic acid bacteria, the use of suitable fermentation strategies is crucial. The growth, and survival of the probiotic bacterium, Lactobacillus paracasei L26, were assessed during a 10-day co-fermentation period with a brewer's yeast, Saccharomyces cerevisiae S-04, in unhopped wort. Isomerized hop extract was added prior to storage of the beers at 25 °C and 5 °C. During co-fermentation in unhopped wort, L. paracasei L26 maintained high viable cell counts above 8 Log CFU/mL, indicating species compatibility with the yeast. The majority of fermentable sugars were attenuated by S. cerevisiae S-04, with a concomitant production of alcohols and esters. Significant amounts of lactic acid were produced by L. paracasei L26 (P < 0.05). During storage with added isomerized hop extract, maximal probiotic viability enhancing effects were observed in the presence of live S. cerevisiae S-04, in combination with refrigeration. The results suggest that beers could be a vehicle for probiotic delivery under appropriate conditions. This was the first study demonstrating the feasibility of utilizing probiotic lactobacilli as starter cultures in beer brewing. •Probiotic lactobacilli are unable to grow and survive in hopped wort.•In unhopped wort, Lactobacillus paracasei could grow when co-fermented with yeast.•Refrigeration and live yeast enhanced probiotic viability during storage with added iso-α-acids.•A novel unfiltered and unpasteurized beer with viable probiotics was developed.