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Objectives: Surveillance of acute respiratory infection (ARI) informs vaccination, preventive, and management decisions. In many countries, immunofluorescence is the cornerstone for ARI surveillance. We aimed to determine the effect of adding multiplex polymerase chain reaction (mPCR) to conventional surveillance in ARI. Methods: Respiratory samples from patients with influenza-like illness (ILI) and severe acute respiratory infection (SARI) were tested by a conventional approach [direct immunofluorescence (DIF) and SARS-CoV-2 PCR, and a subset of samples underwent routine testing]. Negative specimens were tested by multiplex PCR (mPCR), and remain negative samples were sequenced. Descriptive, multivariable regression analyses were conducted. Results: Between March and June 2022, 299 patients were enrolled. Pathogens were detected in 43.8% of samples (131/299) tested by the conventional approach. Of the 168 negatives after the conventional approach, 157 (93.4%) were positive by mPCR, increasing the detection rate to 96.3% (288/299). With the conventional approach, the most frequent pathogen was respiratory syncytial virus (50.3%, 66/131), whereas with mPCR it was Haemophilus influenzae (37.5%, 63/168). mPCR significantly improved pathogen detection in ARI surveillance (Adjusted Incidence Rate Ratios 4.22 95% IC 4.22–5.85). Conclusion: Adding mPCR to respiratory surveillance conventionally based on DIF significantly enhanced virus and bacteria detection. mPCR should be considered for routine ARI surveillance. [ABSTRACT FROM AUTHOR]