The impact of fat content in cheese on eye formation was investigated. Observations in practice have shown that fat-reduced cheeses tend to have more eyes than equivalent full-fat counterparts. A semi-hard cheese with CO2 production through citrate catabolism by Lacticaseibacillus paracasei and a hard Swiss-type cheese with CO2 production by Propionibacterium freudenreichii were produced with different fat content. Four different fat-in-dry-matter levels (∼100 to ∼480 g kg−1) were applied to the semi-hard cheeses and three (∼330 to ∼560 g kg−1) to the hard cheeses. The direct influence of the fat content on eye formation was distinguished from the consequentially altered cheese composition on bacterial fermentation (i.e., on CO2-production). An increasing fat content had a significant (p <0.05) inhibitory effect on relative eye volume in semi-hard and hard cheeses by increasing the capacity of the cheese matrix to solubilise more CO2.