Limosilactobacillus reuteri is a heterofermentative bacterium commonly found in humans. Under anaerobic conditions and in glycerol- containing medium, L. reuteri can produce reuterin through enzymatic reaction. Reuterin has antimicrobial characteristics, such as antifungal activity and spectrum of activity against Gram-positive and negative bacteria, therefore is used for food preservation. Film-forming solutions constituted of polysaccharides and glycerol are also employed, however, reuterin synthesis in these solutions has not yet been reported. The aim of this study was to optimize the in situ reuterin production by L. reuteri DSM 20016 in alginate film-forming solution. The solutions were obtained using alginate (20 g/L) and two independent variables were studied: glycerol (0–300 mmol/L) and L. reuteri initial biomass (≅6, 7, and 8 log CFU/mL). The samples were analyzed every 24h for 72h of anaerobic fermentation (37 °C). The highest reuterin production was 68.39 mmol/L. In general, higher bioconversion rates from glycerol to reuterin were obtained using 50 and 100 mmol/L of glycerol, the concentrations of 200 and 300 mmol/L were not considered effective. High glycerol concentrations during catalysis can inactivate the glycerol dehydratase and consequently limit the reuterin production. The fermentation time did not have a significant effect on reuterin production (p>0.05), which even showed a decrease trend from 24 to 72h of fermentation. This decrease may be due to reuterin interaction with the cellular material and free amino groups in the medium. In addition, the use of L. reuteri initial biomass at approximately 8 log CFU/mL improved the reuterin production and decreased the fermentation time to obtain a higher metabolite concentration. In conclusion, was developed an efficient process for in situ reuterin production in alginate film-forming solution, in which the L. reuteri initial biomass at 8 log CFU/mL and 100 mmol/L of glycerol increased the metabolite production immediately after 24h of fermentation.