Microsporidia are eukaryotic obligatory intracellular parasites that infect a wide range of vertebrates and invertebrate hosts. Spores infect target cells of the host by transferring their sporoplasm through a distinctive polar tube. This study investigated how selected chemicals influence the germination of two newly discovered microsporidia species from central-western Iran. Spores of Parathelohania iranica were extracted from infected larvae of Anopheles superpictus s.l. and purified by the Percoll discontinuous density gradient method. Because of the small number of spores per copepod, extraction and purification were not performed for spores of the microsporidium infecting Paracyclops chiltoni. Both spores were exposed to KCl, NaCl, KI, NaI, and H₂O₂ and the effects of concentration (0.5, 1.5, and 2.5 M), pH (7.0, 9.0, and 11.0), temperature (4 °C and 25 °C), and duration of exposure (10 and 30 min) on spore germination were investigated and compared. This study indicated that the type of the ionic nature of the surrounding environment of spores plays an important role in the release of polar tubes of both microsporidia. Additionally, anions played a more significant role than cations. This effect was directly related to concentration, temperature, and time. However, no specific pattern was recognized at different alkaline pH levels. Hydrogen peroxide was not effective in releasing the polar tubes of the spores of these microsporidia. This study demonstrated the comparative role of some chemicals and the associated factors in the release of the polar tube of two aquatic microsporidia. Future research should examine the practical value of these findings in the mass production of candidate microsporidia for the biological control of pest invertebrate hosts.