This chapter aims to provide an overview on the pharmacology of cannabinoids and compounds targeting the endocannabinoid (eCB) system in preclinical tests for studying anxiolytic drugs. Δ9-tetrahydrocannabinol (THC), the main active compound of Cannabis sativa, induces biphasic responses consisting of anxiolytic- or anxiogenic-like effects at low or high doses, respectively. In addition to THC, cannabinoid type-1 receptor (CB1R) synthetic agonists, with various degrees of affinity and efficacy also affect anxiety-like responses in complex ways, holding limited therapeutic potential. In contrast, the phytocannabinoid cannabidiol exerts consistent anxiolytic-like effects with a favorable side-effect profile; however, its precise mechanism of action remains uncertain. eCB hydrolysis inhibitors take advantage of the on-demand functioning of the eCB system to facilitate CB1R signaling without potential side effects associated with direct agonists. Finally, the interactions between anandamide and other non-canonical components of the eCB system, such as the transient receptor potential vanilloid type-1 channel (TRPV1) have also been investigated, suggesting compounds that simultaneously increase brain eCB levels and block TRPV1 as new pharmacological strategies to alleviate anxiety.