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Optical and spectroscopic methods for biofilm examination and monitoring are reviewed. Biofilm examination techniques include microscopic methods, coupled with image analysis and with oligonucleotide ribosomal RNA probing methods (fluorescence in situ hybridization). Microscopic examination techniques are especially advantageous in extracting biofilm structural and architectural parameters, as well as structure-function relationships of the biofilm microbial population. Spectroscopic techniques are able to elicit biofilm chemical and metabolic patterns, as well as biofilm activity. They are of outstanding importance for on-line, non-invasive biofilm monitoring, especially when coupled with chemometric algorithms for spectra calibration and pattern recognition. The paper emphasises the importance of the combination of novel and established analytical techniques, as well as their integration with powerful computational methods for the automation of biofilm monitoring.