Exacerbations of allergic asthma are often triggered by respiratory viral infections. We have previously shown that in a T-helper type 2 (Th2)-biased cytokine environment, mouse and human airway epithelial cells (AEC) exhibit increased expression of pro-inflammatory and anti-viral genes in response to synthetic double-stranded ribonucleic acid (dsRNA), a virus-like stimulus. This implies coordinated regulation of gene expression, suggesting possible involvement of microRNA. To investigate this, we developed a novel approach to identifying candidate microRNA using online databases, then confirmed their expression by quantitative real-time polymerase chain reaction (qRT-PCR). Using a list of genes of interest, defined on the basis of the previous study as being up-regulated in a Th2 environment, we searched mouse and human microRNA databases for possible regulatory microRNA, and selected 10 candidates that were conserved across species or predicted by more than one human database. Expression of these microRNA was tested by qRT-PCR, in primary human AEC pre-treated with Th2 cytokines and exposed to dsRNA. Expression of hsa-miR-139-5p, miR-423-5p and miR-542-3p was significantly decreased in Th2 pre-treated AEC, and miR-135a-5p exhibited a trend towards decreased expression. Further database searches confirmed that these microRNA regulated additional pro-inflammatory and anti-viral response genes for which expression had previously been shown to be up-regulated, confirming the validity of this approach. Our study demonstrates the value of using multiple online databases to identify candidate regulatory microRNA and provides the first evidence that in an allergic environment, microRNA may be important in altering the pro-inflammatory and anti-viral responses of human AEC during exacerbations of asthma.