Production costs of bacterial cellulose (BC) can be reduced using alternative fermentation media, e. g., various agricultural by-products including whey. This study focuses on whey as an alternative growth medium for BC production by Komagataeibacter rhaeticus MSCL 1463. It was shown that the highest BC production on whey was 1.95 ± 0.15 g/L, which is approximately 40-50% lower that BC production on standard HS media with glucose. It was also confirmed that K. rhaeticus MSCL 1463 can utilise both lactose and galactose as the sole C source in the modified HS medium. Different whey pre-treatment methods showed that the highest BC synthesis with K. rhaeticus MSCL 1463 was achieved in undiluted whey after standard pre-treatment procedure. Moreover, BC yield from substrate on whey was significantly higher (34.33 ± 1.21%) compared to the HS medium (16.56 ± 0.64%), which shows that whey can be used as a potential fermentation medium for BC production.