Enterocytozoon bieneusi and Blastocystis sp. are common zoonotic pathogens that parasitize in the small intestine of humans and animals, posing a threat to public health. However, little information is available on the prevalence and genotypes/subtypes of E. bieneusi and Blastocystis sp. in cattle in Jiangxi Province, southeastern China. In the present study, 556 fecal samples of cattle were collected from Nanchang city, Gao'an city, Xinyu city, and Ji'an city in Jiangxi Province. All samples were examined for the presence of E. bieneusi by nested PCR analysis of the ribosomal internal transcribed spacer (ITS) and Blastocystis sp. using PCR targeting the SSU rRNA gene. The overall prevalence of E. bieneusi and Blastocystis sp. was 5.4% (30/556) and 54.9% (305/556), respectively. The prevalence of E. bieneusi in dairy cattle, beef cattle, and buffaloes was 7.9% (13/165), 3.9% (11/283), and 5.6% (6/108), respectively. Eleven E. bieneusi genotypes were identified in this study, including six known genotypes, D (n = 10), I (n = 5), J (n = 4), IV (n = 4), N (n = 1), and BEB4 (n = 1), and five novel genotypes, JX-I to JX-V (n = 1), with genotype D as the predominant genotype in cattle. Phylogenetic analysis showed that six genotypes of E. bieneusi, D, IV, and JX-II to JX-V, were clustered into zoonotic group 1, whereas the remaining five genotypes belonged to group 2. Moreover, seven, seven, four, and five types were identified by multilocus sequence typing (MLST) at the MS1, MS3, MS4, and MS7 loci, respectively, forming three distinct multilocus genotypes (MLGs). In addition, the prevalence of Blastocystis sp. was 42.4% (70/165), 59.4% (168/283), and 62.0% (67/108) in dairy cattle, beef cattle, and buffaloes, respectively. Sequence analysis revealed that ST1, ST5, ST10, and ST14 of Blastocystis sp. were identified in these cattle, with ST10 being the major subtype. ST1 and ST5 are potential zoonotic subtypes. These findings have important implications for the control of E. bieneusi and Blastocystis sp. in cattle in Jiangxi Province.