Mycoplasma pneumoniae is a novel, cell-wall-less bacterial pathogen of the human respiratory tract that accounts for up to 20% of community-acquired pneumonia. Detection and diagnosis of mycoplasma infections is limited by several factors, including poor success at culture from clinical samples. At present the most effective means for detection and genotyping is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity and specificity but requires separate tests for detection and genotyping, lacks standardization between available tests, and has limited practicality for widespread, point-of-care use. We have developed and previously described a silver nanorod array-surface enhanced Raman spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae in simulated and true clinical throat swab samples with statistically significant specificity and sensitivity, and the ability to distinguish between reference strains of the two main genotypes of M. pneumoniae. Here we ascertained that differences in sample preparation influence the integrity of mycoplasma cells for NA-SERS analysis, which in turn impacts the resulting spectral signature. Furthermore we established the lower limit of detection by NA-SERS for M. pneumoniae intact-cell sample preparations. Using partial-least squares discriminatory analysis (PLS-DA) of sample spectra, we found that NA-SERS consistently detected intact M. pneumoniae to 0.66 genome equivalents (cells/l) with 90% cross-validated statistical accuracy. By comparison, qPCR of samples in parallel yielded a lower limit of detection of 2.5 cells/l. In addition, we used PLS-DA to demonstrate that NA-SERS was able to detect 30 M. pneumoniae clinical isolates from globally diverse origins and M. pneumoniae reference strain controls, and could distinguish all M. pneumoniae clinical isolates and reference strains from a panel of 12 other human commensal and pathogenic Mollicutes species with 100% cross-validated statistical accuracy. Lastly, PLS-DA correctly classified by genotype all 30 clinical isolates with 96% cross-validated accuracy for type 1 strains, 98% cross-validated accuracy for type 2 strains, and 90% cross-validated accuracy for type 2V strains.