Background and objective: Malignant pleural effusion (MPE) affects >90% of mesothelioma patients. Research on MPE has focused on its physical impact on breathlessness; MPE is rich in growth mediators but its contribution to tumour biology has not been investigated. We aimed to examine the potential effects of MPE in promoting growth, migration and chemo-resistance of mesothelioma. Methods: Pleural fluid samples from 151 patients (56 mesothelioma, 60 metastatic pleural cancer and 35 benign) were used. Seven validated human mesothelioma cell lines and three primary cultured mesothelioma lines were employed. Results: Pleural fluid from mesothelioma patients (diluted to 30%) consistently stimulated cell proliferation (trypan-blue cell viability assay) in five mesothelioma cell lines tested by (median) 2.23-fold over controls (all P < 0.0001). The fluid also induced cell migration by (median) 2.13-fold in six mesothelioma cell lines using scratch-wound assay. In a murine flank model of mesothelioma, tumour infused with daily instillations of pleural fluid grew significantly faster over saline controls (median 52.5 cm2 vs 28.0 cm2 at day 13, P = 0.028). Addition of MPE (diluted to 30%) to culture media significantly protected mesothelioma from cisplatin/pemetrexed-induced cell death in all three cell lines tested (median fold reduction of 1.29, 1.98 and 3.90, all P < 0.001 vs control). The growth effects of matched pleural fluid and cultured mesothelioma cells from the same patients did not differ significantly from unmatched pairs. Conclusion: This ‘proof-of-concept’ study reveals potent biological capabilities of malignant pleural fluid in mesothelioma pathobiology.