Blastocystis is a common enteric protist that is linked to intestinal and extra-intestinal diseases. At least 24 subtypes (STs) have been described, with the main colonization of ST1–ST4 in humans. In our attempt to determine the distribution of Blastocystis STs in Olsztyn and surroundings in northeastern Poland, 319 stool samples from volunteers were subjected to copro-ELISA and PCR testing. Positive findings were identified in 77, 48, and 46 of the samples via copro-ELISA, PCR, and sequencing, respectively. Blastocystis colonization was not associated with gender or dwelling place but was statistically higher in people age 60–69 yr (32.6%). Five STs (ST1–ST4, ST7) were identified, in which ST3 (37%) was most prevalent, followed by ST2 (19.6%), ST1 (17.4%), ST4 (13%), and ST7 (8.7%). The current study revealed a similar rate of microorganism colonization in Polish volunteers compared to other developed countries, without significant differences in gender and dwelling place. Significant statistical differences were found in different age groups, where Blastocystis was highly detected in elderly people. In the current study, PCR was the most plausible method based on the sequencing results.