Studies using the embryos of the freshwater prawn Macrobrachium olfersii have reported changes in embryonic cells after exposure to ultraviolet B (UVB) radiation, such as DNA damage and apoptosis activation. Considering the importance of mitochondria in embryonic cells, this study aimed to characterize the aspects of mitochondrial morphofunctionality in M. olfersii embryos and mitochondrial responses to UVB radiation exposure. The coding sequences of genes Tfam, Nrf1, Mfn1, and Drp1 were identified from the transcriptome of M. olfersii embryos. The phylogenetic relationship showed strong amino acid identity and a highly conserved nature of the sequences. Additionally, the number of mitochondrial DNA (mtDNA) copies were higher in the early embryonic days. The results showed that the expression of the analyzed genes was highly regulated during embryonic development, increasing their levels near hatching. Furthermore, when embryos were exposed to UVB radiation, mitochondrial biogenesis was activated, recognized by higher levels of transcripts of genes Tfam and Nrf1, accompanied by mitochondrial fission. Additionally, these mitochondrial events were supported by an increase of mtDNA copies. Our results showed that UVB radiation was able to change the mitochondrial morphofunctionality, and under the current knowledge, certainly compromise embryonic cellular integrity. Additionally, mitochondria is an important cellular target of this radiation and its responses can be used to assess environmental stress caused by UVB radiation in embryos of aquatic species.