Washingtonia filifera is important for its ecological, economic, cultural, horticultural, ornamental, and medicinal potential. Elicitation of in vitro cultures presents a promising and efficient method for the large-scale production of valuable bioactive compounds. This study assessed the effect of chitosan concentration (0, 20, 40, 60, 80, 100 mg L⁻¹) on biomass growth [fresh weight (FW), dry weight (DW)] and phytochemical profile [total phenolic content (TPC), total flavonoid content (TFC), DPPH antioxidant activity, total phenolic productivity (TPP), total flavonoid productivity (TFP)] in W. filifera callus and cell suspension cultures. Among different plant growth regulator combinations tested, 3 mg L⁻¹ 2,4-D + 0.5 mg L⁻¹ 2ip gave higher callus induction (90%) (MS medium, 12 weeks). A maximum growth curve (FW: 180 mg) of cell suspension culture was achieved 7 weeks after initiation (shaker at 90 rpm for 24 h). Cell suspension exhibited higher FW, DW, TPC, TFC, DPPH, TPP, and TFP than callus, while flavonoid production was higher than phenolic production. FW and DW were higher in both systems, with 40 mg L⁻¹ chitosan. Chitosan at 60 mg L⁻¹ best enhanced the phytochemical profile of both the 4-week solidified callus and the 7-week liquid cell suspension (TPC: 29.9 and 32.1 mg GAE g⁻¹ DW; TFC: 40.5 and 56.1 mg QE g⁻¹ DW; TPP: 969.2 and 1122.6 mg L⁻¹; TFP: 1313.9 and 1521.7 mg L⁻¹; DPPH: 87.4 and 92.3%), respectively, while 40 mg L⁻¹ chitosan was equally effective regarding DW, TFC, and TFP in cell suspension. Chitosan elicitation provides a powerful strategy to upregulate phenolic and flavonoid biosynthesis in W. filifera in vitro systems, conferring superior antioxidant potential. The identification of peak elicitation parameters (chitosan concentration, exposure time) allows for the targeted enhancement of bioactive compound yields, suggesting a viable path for industrial bioproduction and commercialization in pharmaceuticals, nutraceuticals, and functional foods, leveraging bioreactor technology for efficient scale-up.