Historically, humans have been using Cannabis sativa for both recreational and medical purposes. Nowadays, cannabis-based products have gained scientific interest due to their beneficial effects on several syndromes and illnesses. The biological activity of cannabinoids is essentially due to the interaction with the endocannabinoid system, and zebrafish (Danio rerio) is a very well-known and powerful in vivo model for studying such specific interactions. The aim of the study was to investigate the effects of different doses of a Cannabis sativa whole extract [dissolved in dimethyl sulfoxide (DMSO)] on zebrafish eggs’ hatchability, embryo post-hatching survival, larvae locomotion behavior and mRNA gene expression. The results showed the absence of toxicity, and no significant differences were observed between treatments for both embryo hatching and survival rate. In addition, larvae exposed to the cannabis extract at the highest dose [containing 1.73 nM and 22.3 nM of ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively] showed an increased locomotion compared to the control and DMSO treated groups. Moreover, qRT-PCR analysis showed that the highest dosage of cannabis induced an over-expression of cnr1 and cnr2 cannabinoid receptors. In conclusion, the exposition of zebrafish larvae to the whole extract of Cannabis sativa showed no negative effects on embryo development and survival and enhanced the larvae’s locomotor performances. These findings may open up possible Cannabis sativa applications in human pharmacology as well as in other animal sectors.