The encapsulation of the Pimenta dioica (P. dioica) essential oil in chitosan and chitosan/k-carrageenan microspheres was investigated. The P. dioica essential oil was extracted from P. dioica (L) Merr. berries by means of supercritical CO2. 23 components, among which eugenol (68.06%), methyl eugenol (9.37%), were identified through GC analysis. The study of the antioxidant activity revealed that the IC50 (DPPH) value for the P. dioica essential oil is significantly lower (p ≤ 0.05) than the value of the butylated hydroxytoluene (BHT) and of the ascorbic acid (AAc). The microspheres containing P. dioica essential oil were prepared through the extrusion of the oil-in-water (O/W) emulsions using as wall material the chitosan and the chitosan/k-carrageenan in different mass ratios. Chitosan microspheres present a high swelling degree than the chitosan/k-carrageenan microspheres (p < 0.05). The release kinetics of the P. dioica essential oil presented a case II transport mechanism (zero order release kinetics) for chitosan/k-carrageenan microspheres (1:1 mass ratio) and a non-Fickian release mechanism for chitosan/k-carrageenan microspheres (1:0, 3:1 and 2:1). The morphology of the microspheres, the efficiency of the encapsulation and the antimicrobial activity of the P. dioica essential oil crude and microencapsulated essential oil were studied. Microencapsulated P. dioica essential oil exhibited antimicrobial activity against Candida utilis, Bacillus cereus and Bacillus subtilis.