For centuries, bromelain has been used to treat a range of ailments, even though its mechanism of action is not fully understood. Its therapeutic benefits include enzymatic debridement of the necrotic tissues of ulcers and burn wounds, besides anti-inflammatory, anti-tumor, and antioxidant properties. However, the protease is unstable and susceptible to self-hydrolysis over time. To overcome the stability issues of bromelain, a previous study formulated chitosan-bromelain nanoparticles (C-B-NP). We evaluated the optimized nanoformulation for in vitro antioxidant, cell antiproliferative activities and cell migration/proliferation in the scratch assay, comparing it with free bromelain. The antioxidant activity of free bromelain was concentration and time-dependent; after encapsulation, the activity level dropped, probably due to the slow release of protein from the nanoparticles. In vitro antiproliferative activity was observed in six tumor cell lines for free protein after 48 h of treatment (glioma, breast, ovarian, prostate, colon adenocarcinoma and chronic myeloid leukemia), but not for keratinocyte cells, enabling its use as an active topical treatment. In turn, C-B-NP only inhibited one cell line (chronic myeloid leukemia) and required higher concentrations for inhibition. After 144 h treatment of glioma cells with C-B-NP, growth inhibition was equivalent to that promoted by the free protein. This last result confirmed the delayed-release kinetics of the optimized formulation and bromelain integrity. Finally, a scratch assay with keratinocyte cells showed that C-B-NP achieved more than 90% wound retraction after 24 h, compared to no retraction with the free bromelain. Therefore, nanoencapsulation of bromelain with chitosan conferred physical protection, delayed release, and wound retraction activity to the formulation, properties that favor topical formulations with a modified release. In addition, the promising results with the glioma cell line point to further studies of C-B-NP for anti-tumor treatments.